Rapid quantitation of specific analytes in an individual's blood is critically important for the diagnosis of disease and its severity, often under emergency conditions, in the monitoring of the progression of pathological conditions and following the recovery process brought about by surgical and drug therapies. It is often important to know not only whether a specific analyte is present, but as well its level, in order to determine the present stage of a particular condition or disease in order to prescribe the most effective remedy at that particular stage. In the treatment of many diseases, a particular therapy may be ineffective or toxic if given at the wrong stage of the condition. For example, the levels of specific markers of cardiac muscle damage and the relationship among them may indicate that a patient has had or may be having a heart attack. The level of a therapeutic drug in the circulation may indicate whether the patient is being dosed optimally, and whether presumptive side effects are indeed due to excess levels of the drug. In infection and sepsis, the circulating levels of infectious microorganism toxins and inflammatory mediators produced by the patient's white blood cells may indicate the severity and level or stage of sepsis and help identify the most efficacious course of therapy. Quantitation of analytes under emergency conditions and using this information to prescribe a particular therapy may mean the difference between saving a patient's life and contributing to the patient's death.
For example, in the case of infection, hospital and particularly intensive care unit patients who have acquired nosocomial infections as a result of peri- or post-operative immunosuppression or secondary to other disease processes, such as pancreatitis, hypotensive or hypovolemic shock, physical trauma, burn injury, or organ transplantation, and develop septic shock syndrome have a mortality which has been quoted to range from 30-70% depending upon other co-incident complications. Despite the development of increasingly potent antimicrobial agents, the incidence of nosocomial infections and, in particular, infections leading to sepsis or septicemia is increasing. The difficulty with many of the promising therapeutic agents is that their window of opportunity and indications for use have not been adequately delineated largely due to a lack of appropriate rapid and quantitative diagnostic procedures and partly due to a lack of complete understanding of the pathogenesis of the sepsis syndrome.
As described in co-pending applications Ser. Nos. 08/552,145 and 08/516,204, both incorporated herein by reference, the presence of bacteria, viruses or fungi or their cell wall components including gram-positive peptidoglycans, lipoteichoic and teichoic acids, and gram-negative endotoxin (lipopolysaccharide, LPS) in blood is indicative of an infection. In addition, the immume system's reaction to the presence of these foreign antigens by the production of pro-inflammatory cytokine mediators such as interleukin-1 (IL-1), tumor necrosis factor (TNF) and interleukin-6 (IL-6), is also indicative of an infection. The quantity of these analytes in circulation may be used to indicate the severity and level or stage of sepsis. For instance, at an early stage of Gram-negative sepsis, LPS may be present at a concentration as low as 5 pg/ml of whole blood. At the next stage, sepsis has progressed and a mediator of sepsis, TNF, can be detected and measured using antibody against TNF. At stage 3, TNF may be present in smaller amounts since it is transitory and another transitory mediator. IL-1, may appear. As sepsis progresses further. LPS levels may decrease and TNF be absent, but IL-1 may increase and interleukin-6 (IL-6) may appear. Finally, in a more prolonged case of sepsis, LPS may be present and IL-1 may be at low levels but IL-6 may be at very high levels. Thus, diagnosis of sepsis and identifying its stage in the course the disease are critical for the successful treatment of this serious and potentially lethal consequence of infection. Quantitation of the levels of the sepsis-associated analytes provide information necessary to determine the best course of therapy to treat the acute disease.
Until the recent advent of novel therapeutic strategies, sepsis patients have been managed largely by palliative care and administration of antibiotics. The biotechnology industry has facilitated the large scale production of many new targeted biopharmaceuticals which utilize monoclonal antibodies against such initiators of sepsis as gram-negative endotoxin (Centocor's HA-1A(R) or Xoma's Xomen-E5(R)), tumor necrosis factor (various producers including Hoffman La Roche and Centocor with patents WO 90/06514 and WO 92/16553), interleukins, as well as various soluble receptor antagonists such as IL-1 RA (Synergen) and sCR.sub.1 (soluble complement receptor 1)- a truncated recombinant complement regulatory molecule. The cost of these therapeutic agents is significant, being priced at $3,000.00 to $4,000.00 per dose. Thus providing this therapy indiscriminately to patients would add a considerable burden to the health care system without providing a corresponding benefit to patients. In addition, there is need for means to monitor the efficacy of such novel therapies.
Currently, one of the major problems with many of the therapeutic protocols being tested by the pharmaceutical companies conducting clinical trials in sepsis intervention is their inability to rapidly detect early and evolving sepsis. The results of blood cultures may arrive too late. Other septicaemia tests are also time consuming and may not be sensitive enough for early detection. Centocor Inc.'s immunometric assay for tumor necrosis factor-alpha (TNF-.alpha.), as described in WO 90/06314, uses two antibodies, one of which is labeled. The National Aeronautics and Space Administration detects Pseudomonas bacteria by extraction of Azurin and detection using Azurin-specific antibody (U.S. Pat. No. 7,501,908). The endotoxin assay kit from Bio Whittaker (Walkerville. Md., U.S.A.) or Seikagaku Kogyo Ltd. (Tokyo, Japan) is a Limulus Amebocyte Lysate (LAL) Assay technique which may be used as a comparison for the present invention.
Many investigators versed in the complexities of the septic response believe that treatment is ineffectual for patients who already manifest the classical clinical symptoms of sepsis (i.e., hyperdynamic circulation, hypotension, decreased systemic vascular resistance, pyrexia and increased oxygen dependency). The course of the inflammatory process has progressed too far for many of the interventions to benefit the patient since the multiple interacting inflammatory cascades with which the body attempts to eliminate the infectious challenge are in many instances at their nadir and difficult to control pharmacologically. Thus, a major clinical and diagnostic challenge is to identify and stage patients, ideally early in the progression of the septic response, or to identity those patients at high risk of developing fulminant sepsis syndrome. The same therapeutic agents given at the one stage in the septic process may have more significant beneficial effects than when given at another, since it is clear that an optimal window period may exist for the efficacy of any particular therapeutic agent. For example, giving a patient antibodies or receptors directed against gram-negative endotoxins when the patient has no detectable levels of these agents present in the circulation and already has a maximally activated cytokine cascade is a waste of resources and of no benefit to the therapy of the patient. The potential market for these anti-sepsis strategies remains large (about 250,000 cases per year in the USA) and has been limited by the inability to identify and stage patients who could benefit from the appropriate pharmacologic interventions.
In addition to infection and sepsis, the diagnosis of many other diseases and conditions are contemplated in the present invention, as their respective diagnostically-useful analytes in circulation can be quantitated by the method of the present invention. Circulating levels of analytes such as hormones, acute phase proteins, toxins, drugs of abuse, markers of cardiac muscle damage, therapeutic drug levels, cytokines and chemokines are among the many clinically useful markers of interest to physicians and other health care professionals, even in an in-home setting, for the diagnosis of and monitoring the treatment of health and disease.
Copending applications Ser. Nos. 08/552,145 and 08/516,204 describe methods to indicate the presence of a preselected analyte in a blood sample by first forming an immunocomplex between the preselected analyte and an antibody to the preselected analyte which is added to the sample. Sepsis-associated analytes such as endotoxin are used as examples. Any immunocomplex formed as a result then activates complement present in the blood sample which in turn causes neutrophils and other white blood cells present in the blood sample to produce oxidants (see FIG. 1). The oxidants then cause an added chemiluminogenic compound, such as luminol, to release light energy. The white blood cells can be optionally additionally stimulated with the addition of opsonized zymosan or other agents resulting in increased production of oxidants. The amount of light emitted over time can be measured by a luminometer device to indicate the presence of analyte in the sample. A control sample without the addition of antibody may be included, to which may also be added any additional stimulatory agents. This method provides a semi-quantitative determination of the level of the pre-selected analyte in the blood sample.
The present invention is an improvement over that described in the co-pending applications which permits the quantitation of the analyte in the blood sample. The present invention takes advantage of the same method as described in the co-pending application, but to achieve a quantitative determination of the analyte, an additional measurement is made of the maximal response of the patient's white blood cells to immunocomplexes, providing a value to which the chemiluminescence produced by immunocomplexes formed from the preselected analyte is compared. This additional measurement provides the necessary information to render the method of the present invention quantitative and permits the staging of sepsis.